Diphtheria is the prototype of an exotoxin-mediated infectious disease. Diphtheria toxin acts on toxin-sensitive cells by: 1) binding, via its B-fragment, to specific cell surface receptors, 2) translocation of the A-fragment into the cytoplasm, and 3) inhibition of protein synthesis by the fragment A-catalyzed ADP-ribosylation of elongation factor 2. The overall objective of this proposal is to characterize the receptor-binding domain(s) of the toxin molecule, and to fully characterize the specific diphtheria toxin-binding cell surface glycoproteins and their toxin-binding domain(s). The diphtheria toxin-binding glycoproteins are detected utilizing a glycoprotein-enriched fraction prepared from exogenously radiolabeled cells, followed by immunoprecipitation with diphtheria toxin plus antiserum to diphtheria toxin. By this method one major diphtheria toxin-binding glycoprotein is detected in hamster thymocytes while various (up to five) diphtheria toxin-binding glycoproteins are detected on the cell surface of toxin-sensitive cells grown in tissue culture (e.g. Vero, baby hamster kidney and Chinese hamster ovary cells). The nature of this multiple diphtheria toxin-binding glycoproteins will be investigated by both biochemical and immunological approaches. Specific monoclonal antibodies will be prepared against each of these glycoproteins and will be tested for their protective effect against toxin-mediated cytotoxicity in order to determine which one of the diphtheria toxin-binding glycoproteins represents the true physiological toxin receptor on cells. These antibodies will also be utilized to investigate the cell surface distribution and the internalization mechanism of the diphtheria toxin-binding glycoproteins. The results of this proposed research will significantly extend our knowledge of diphtheria toxin: receptor interactions and will further our understanding of the mechanisms involved in receptor-mediated internalization of such macromolecules as hormones, growth factors, and other exotoxins.